Project summary Photoreceptor death due to retinal degenerations is the leading cause of blindness in the developed world. Recent approaches to treat these blinding diseases include re-conferring the light sensitivity of the degenerated retina, either by genetic manipulation or stem-cell transplants of photoreceptors. The fundamental impediment to translating these treatments to the living human retina is the ability to visualize and manipulate their functional mechanisms at a cellular scale in vivo. Efforts to functionally probe photoreceptor physiology have relied mainly on bulk measures (electroretinograms or ERG) in vivo, while finer spatial scales are accessible only ex vivo via electrophysiology (patch clamp or suction-electrode recordings) and are thus unsuitable for clinical use. We propose a novel approach rooted in classical interferometry to record how photoreceptors interact with light on a cellular scale. The transduction of photons to electrical signals is well- characterized by a set of biochemical molecular events in cones and is also accompanied with a change in physical structure at nm/ms spatiotemporal resolution. These physical changes can be reliably encoded in the phase of the interferometric signal emanating from the photoreceptors when light of low coherence is used to image them. In Aim 1, we will validate a parallel, phase-resolved optical coherence tomography system(OCT) ? essentially a low-coherence interferometer ? to image the light-driven optophysiology activity in cones. We have implemented a free-space OCT system that is based on line-field, spectral domain operation. This allows an entire phase-stable B-scan to be obtained in a single camera snapshot allowing the probing of multiple cells in parallel. Further, with the use of high-speed aerial sensors, extremely fast volume rates are obtainable. We will first validate our approach by measuring the light-driven optical activity in zebrafish photoreceptors in vivo and compare them against Gnat2-/- mutants which do not exhibit the traditional phototransduction cascade. These measurements will categorize the light-driven optophysiological signal on the basis of those arising from light-opsin interaction vs. those arising due to phototransduction. In Aim 2, we will translate the OCT platform to living humans by combining it with an existing adaptive optics scanning laser ophthalmoscope (AOSLO), such that cone activity can be monitored by individually stimulating them in isolation. A power spectrum detailing the dependence of phase sensitivity on eye motion will be estimated by obtaining high fidelity motion traces from the AOSLO. The application of this technology will be validated in humans by probing a few hallmarks of the trichromatic cone mosaic - spectral topography & spectral sensitivity - and comparing them against retinal densitometry and single-unit recordings respectively. These multiple stages of validation will set the stage for a wider application of this technology to clinical and basic research thus potentially transforming mechanistic studies of retinal circuitry and its diseases.